Schizophrenia is a frequent psychotic disorder leading to severe human suffering and increased suicide rates. Quality and severity of disease phenotypes vary dramatically between patients and the pathogenetic mechanisms on the molecular, cell biological, or neuronal circuit level are poorly understood. However, human genetics studies have demonstrated that genetic factors contribute to the highest risk factors to develop disease. The importance of genetic factors for disease development is widely accepted in the field. Because of our ability to manipulate and define genetic backgrounds and specific factors (as opposed to potential environmental factors) in cultured cells, it is possible that critical disease traits of the human brain can be recapitulated in cultured cell-based models. Recent advances by us and others in the field of epigenetic reprogramming and stem cell biology has made it possible to generate fully functional human neurons from pluripotent stem cells. We are therefore very close to interrogate human disease neurons for abnormal cell- biological traits in a meaningful way. Projects 1 and 2 will begin to analyze disease-specific traits using existing methods. However, current technology has several shortcomings limiting the full phenotypic characterization. The goal of Project 3 (this project) is to further develop and optimize existing stem cell methods that will allow to substantially increase the spectrum of assays to be analyzed. As the protocols are being developed and become available in Project 3, they will be immediately implemented and utilized in Projects 1 and 2. In particular, Project 3 will develop two critical components for the overall consortium grant: (1) It will provide the tools to genetically engineer conditional and/or definitive single gene and large CNV mutations. (2) The project will develop methods to develop defined inhibitory neuronal subtypes. Together with our already existing method to generate pure excitatory neuronal subtypes this will allow us to generate mixed cultures with defined excitatory/ inhibitory neuronal components which will allow the characterization of inhibitory synaptic transmission. In addition, the project will work on two non-essential but highly desired protocol developments: (1) Methods will be devised for industrial generation of neurons in large scale to improve consistent phenotypic analyses and enable the establishment of human cell models for pharmaceutical drug development. (2) We will develop ways to eliminate the currently required co-culture of mouse glia to be replaced with defined substances or human cells because use of cell models containing supportive mouse cells may complicated therapeutic drug development for human use in vivo. We believe the proposed technology developments will be critical contributions to the field and ultimately enable the generation of authentic human disease cell models.